Abnormalities that occur during neural crest cell (NCC) development are directly responsible for many human congenital and hereditary malformations, diseases and cancers. Premigratory NCCs exist as adherent epithelial cells in the embryonic dorsal neural tube but later become motile through an epithelial-to- mesenchymal transition (EMT) in response to numerous intrinsic and extrinsic cues. Importantly, this event is analogous in scope to abnormal EMTs that underscore human disease and cancer. Although several similarities exist between these normal and aberrant EMTs, the chief amongst these is the cascade of events initiated by loss of cell surface cadherin proteins and the disassembly of adherens junctions to facilitate cell motility. Work from our lab has shown that loss of Cadherin6B (Cad6B) in premigratory cranial NCCs is crucial for NCC EMT. Cadherin reduction in vitro is mediated both transcriptionally and post-translationally through proteolysis and endocytosis. Our prior studies described comparable mechanisms of transcriptional repression and proteolytic processing of Cad6B in vivo during NCC EMT. Our new preliminary data, however, now reveal that Cad6B possesses putative endocytic motifs and co-localizes with markers of endosomes and lysosomes, both in vitro and in vivo. These initial findings suggest that Cad6B is also regulated post-translationally via endocytosis and, to our knowledge, are the first demonstration of cadherin endocytosis during an in vivo EMT. Based on these data, we hypothesize that Cad6B endocytosis plays a critical function during NCC EMT and migration through removal of Cad6B protein from premigratory NCC plasma membranes, thereby promoting the dismantling of adherens junctions and NCC migration. The Specific Aims of this application are to 1) define how mutations in putative endocytic motifs affect Cad6B internalization and 2) assess effects of disruptions in Cad6B endocytosis on NCC EMT and migration. In Aim 1, we will alter specific amino acids within motifs that may regulate Cad6B endocytosis and assess effects on Cad6B internalization, both in vitro and in vivo. In Aim 2, we will evaluate how disruptions in Cad6B endocytosis impact NCC EMT and migration through in vivo and in vitro cell and molecular assays. The proposed research is innovative because it takes a multi-disciplinary approach that combines embryology, biochemistry, and cell and molecular biology to examine post- translational mechanisms of cadherin regulation during a biologically relevant EMT. These studies are significant because the results will enhance our understanding of mechanisms associated with generating migratory cell types during both normal developmental and aberrant EMTs, and could lead to the development of new treatments for human diseases.